This project aims to understand the role and mechanism of function of HIV regulation in the virus life cycle and disease development; and to dissect the mechanisms of nucleocytoplasmic trafficking of macromolecules. The conclusions of this work are applied towards the design of better vaccine approaches against AIDS.[unreadable] [unreadable] The essential viral protein Rev was the first characterized factor to direct the export of mRNA from the nucleus to the cytoplasm. It has been demonstrated that Rev is a valid target for antiviral strategies. The multimerization of Rev protein is crucial for the life cycle of HIV-1. The development and study of Rev-independent HIV and SIV molecular clones has shown that even partial inhibition of Rev function can severely diminish the pathogenic potential of these viruses. Therefore, a drug that inhibits Rev function would be valuable for AIDS therapy as a new class of compounds. The trafficking of Rev, its multimerization and interaction with the nuclear export receptor CRM1 have been studied in live cells using sensitive fluorescent techniques such as fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We had previously developed several strong GFP and BFP mutants used by many investigators. Some of these mutants were ideal for FRET applications. This technology was developed as part of our efforts to use GFP to study gene expression and macromolecular interactions in real time in live cells. An important finding was that Rev forms multimers also in the cytoplasm, which are different than the multimers observed in the nucleolus. We discovered that CRM1 is able to interact with its cargo independently of RanGTP, and we suggested that binding of RanGTP to CRM1 triggers a conformational change, which is consistent with recent findings by other groups. The results advance our understanding of the Rev function and set the stage for detailed analysis of the complexes responsible for Rev-directed mRNA export. The materials generated by these studies also led to the development of rational algorithms measuring the colocalization of proteins in live cells. We have previously identified an extensive family of RNA transport elements (RTE) in the mouse genome able to replace the HIV-1 Rev/RRE posttranscriptional regulatory system, using a mutated HIV-1 DNA proviral clone as a novel molecular trap. This is general methodology for the identification of cis-acting posttranscriptional control elements in the mammalian genome. We have analyzed in detail the structure and function of the RTE element, and have made great progress in the identification of cellular factors participating in this export pathway. These results contribute to the further understanding of the basic mechanisms of nucleocytoplasmic traffic of macromolecules.[unreadable] [unreadable] The understanding of the regulatory mechanisms of HIV gene expression has been applied to the development of improved DNA vaccination approaches. We have developed a general method to increase the expression of unstable mRNAs by introducing multiple point mutations in their coding regions. This results in efficient transport, and increased stability and translation of many mRNAs. Based on our results with gag and env sequences of HIV, we developed efficient expression vectors for DNA-based immunization. These constructs are also used on viral vectors with superior results. We showed that better antigen expression results in increased immunogenicity. We developed additional DNA vaccine vectors producing modified antigens in order to elicit more potent immune responses in primates. We tested several such vectors expressing either secreted or intracellularly degraded antigens and showed that some combinations increase immunogenicity.